Papillomaviruses induce proliferative epithelial lesions and can only undergo vegetative replication in terminally differentiated keratinocytes. This has hampered the study of the complete viral life cycle because of the difficulties in generating a differentiated stratified epithelium in tissue culture. Using a combination of organotypic raft cultures and xenografts on nude mice we have developed a system in which bovine papillomavirus type 1 (BPV-1) can replicate and produce infectious viral particles. Organotypic cultures have been established with bovine keratinocytes plated on a collagen raft containing BPV-1 transformed fibroblasts. These keratinocytes were infected with virus particles isolated from a bovine wart or were transfected with cloned BPV-1 DNA. Several days after lifting the rafts to the air interface, they were grafted on nude mice. Large xenografts were produced that exhibited a hyperplastic and hyperkeratotic epithelium overlying a large dermal fibroma. These lesions were strikingly similar to a fibropapilloma caused by BPV-1 in the natural host. Amplified viral DNA and capsid antigens were detected in the suprabasal cells of the epithelium. Moreover, infectious virus particles could be isolated from these lesions and quantitated by a focus formation assay on mouse cells in culture. Interestingly, analysis of grafts produced with infected and uninfected fibroblasts indicated that the fibroma component was not required for productive infection or morphological changes characteristic of papillomavirus-infected epithelium. This system will be a powerful tool for the genetic analysis of the roles of the viral gene products in the complete viral life cycle.